Over the last 4.5 years of this Merit award (R37 MH63105) we have achieved a number of milestones: (1) the development of a single vesicle fusion assay that mimics certain properties of spontaneous and calcium triggered vesicle fusion observed in neuronal cultures; (2) the atomic resolution structure of the complex between the calcium sensor synaptotagmin-1 and the neuronal SNARE complex, revealing an unexpected calcium-independent interface that we believe forms the foundation for the process of calcium-triggered synaptic vesicle fusion; (3) the near-atomic resolution structure of the complex between NSF, SNAPs, and SNAREs that has revealed clues how NSF is capable of disassembling the SNARE complex. Moreover, we have studied the molecular mechanism of complexin-1 and Munc18. We found that complexin-1 inhibits spontaneous release and activates calcium-triggered vesicle fusion in our synthetic system, consistent with complexin's function as observed in neuronal cultures. However, we found no effect of Munc18 on intrinsic fusion rates, suggesting that its primary function is to facilitate SNARE complex formation. The specific aims for the next 5 year period are as follows: (1) Decipher the molecular mechanism of NSF-mediated SNARE disassembly. We plan to determine structures of NSF and of the complex of NSF, SNAREs and SNAPs upon hydrolyzing ATP by using single- particle cryo-EM. (2) Study how complexin and synaptotagmin-1 cooperate in fast synchronous release. Our recent work revealed a conserved, calcium-independent interface between synaptotagmin-1 and the neuronal SNARE complex. Following up on this result, we plan to investigate the interplay between synaptotagmin-1, and complexin-1, and the neuronal SNARE complex at the atomic level. We plan to crystalize this supercomplex, along with functional studies in neuronal cultures in order to test the new interfaces that we may discover in the crystal structure. (3) Investigate the role of Munc13. We will test the hypothesis that Munc13 is a facilitator for efficient SNARE complex formation. (4) Establish a hybrid fusion assay to study the fusion kinetics of purified endogenous synaptic vesicles obtained from mice brains. We will extend our single vesicle fusion assay to use purified synaptic vesicles in combination with synthetic plasma membrane mimicking ?acceptor? vesicles. We hypothesize that different pools of synaptic vesicles may result in different fusion kinetics.